Project Summary The motivation for my research program has been to understand how oncogenic signaling promotes human cancer. An important facet of our work, which distinguishes our contributions from those of others, is that we have focused on fundamental questions about molecular mechanisms of both normal and aberrant signaling, emphasizing structural, biochemical, molecular, and cell-based approaches. My overarching research goals over the award period build from our sustained contributions over almost two decades in the Notch field, and will emphasize answering the following key questions: i) What is the molecular mechanism of normal and pathogenic Notch activation by ADAM-family proteases? Ligands normally convert Notch receptors from a dormant to signaling-active state by rendering them susceptible to proteolysis by ADAM-family metalloproteases at a site immediately external to the cell membrane, yet our understanding of ADAM-mediated proteolysis of Notch receptors remains remarkably rudimentary. We will elucidate the structures of the full ADAM10 and ADAM17 ectodomains, and determine how interplay between the regulatory and catalytic domains of the ADAMs executes Notch proteolysis in normal and oncogenic signaling. Structural features that distinguish the ADAM10 active site from that of ADAM17 can then be used to guide development of highly selective ADAM10 or ADAM17 inhibitors. ii) What is the molecular mechanism of engagement of the transcriptional machinery by Notch nuclear complexes? Binding of intracellular Notch (ICN) to the RBPJ transcription factor in the nucleus leads to MAML recruitment, but how Mastermind-like proteins (MAMLs) cooperate with Notch-RBPJ complexes to induce transcription remains a fundamental unresolved question in the field. By defining the sequence of events connecting Notch1 signaling and MAML1 recruitment to gene expression changes, we will illuminate the role of MAML in the response of cancer cells to Notch activation. iii) What is the molecular mechanism of feedback regulation by Notch transcriptional targets? As a core transcriptional target regulated by Notch in diverse cell types, NRARP is an integral part of the Notch signal transduction circuitry, acting as a negative feedback regulator of signaling by binding to the core Notch transcription complex. Our goals will be to elucidate the structural basis for NRARP recruitment to the Notch transcription complex, uncover the molecular mechanism underlying assembly of NRARP inhibitory complexes, and elucidate the molecular mechanism underlying NRARP inhibition. Together, these studies will uncover a central mechanism of Notch signal modulation, and create opportunities for development of novel regulators of Notch signaling.